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INSULIN, HUMAN
Insulinum humanum
(2.2.29).
Liquid chromatography
Column :
size : l = 0.10 m, = 4.6 mm,
C257H383N65O77S6
Mr 5808
DEFINITION
Human insulin is a 2-chain peptide having the structure of
the antidiabetic hormone produced by the human pancreas.
Content : 95.0 per cent to 105.0 per cent of human insulin
C257H383N65O77S6 plus A21 desamido human insulin (dried
substance).
By convention, for the purpose of labelling insulin
preparations, 0.0347 mg of human insulin is equivalent to
1 IU of insulin.
PRODUCTION
Human insulin is produced either by enzymatic modification
and suitable purification of insulin obtained from the
pancreas of the pig or by a method based on recombinant
DNA (rDNA) technology.
Human insulin is produced under conditions designed to
minimise the degree of microbial contamination.
For human insulin produced by enzymatic modification
of insulin obtained from the pancreas of the pig, the
manufacturing process is validated to demonstrate
removal of any residual proteolytic activity. The competent
authority may require additional tests.
For human insulin produced by a method based on
rDNA technology, prior to release the following tests are
carried out on each batch of the final bulk product, unless
exemption has been granted by the competent authority.
Host-cell-derived proteins. The limit is approved by the
competent authority.
Single chain precursor. The limit is approved by the
competent authority. Use a suitably sensitive method.
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water and in ethanol
(96 per cent). It dissolves in dilute mineral acids and with
decomposition in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (a).
1800
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 - 60
90 30
10 70
60 - 65
30 0
70 100
65 - 70
100
Insulin, human
TESTS
Impurities with molecular masses greater than that of
insulin. Size-exclusion chromatography (2.2.30) : use the
normalisation procedure.
Test solution. Prepare a solution containing 4 mg/ml of the
substance to be examined in 0.01 M hydrochloric acid.
Resolution solution. Use a solution of insulin
(about 4 mg/ml), containing more than 0.4 per cent
of high molecular mass proteins. An injectable insulin
preparation, whether a solution or a suspension, that has
been clarified with a sufficient amount of 6 M hydrochloric
acid, containing the indicated percentage of high molecular
mass proteins, or a solution prepared from insulin, dissolved
in 0.01 M hydrochloric acid, may be used. Insulin containing
the indicated percentage of high molecular mass proteins
may be prepared by allowing insulin powder to stand at room
temperature for about 10 days.
Maintain the solutions at 2-8 C and use within 7 days. If
an automatic injector is used, maintain the temperature
at 2-8 C.
Column :
size : l = 0.3 m, = minimum 7.5 mm,
stationary phase: hydrophilic silica gel for
chromatography R (5-10 m) with a pore size of
12-12.5 nm, of a grade suitable for the separation of
insulin monomer from dimer and polymers.
Mobile phase : mix 15 volumes of glacial acetic acid R,
20 volumes of acetonitrile R and 65 volumes of a 1.0 g/l
solution of arginine R ; filter and degas.
Flow rate : 0.5 ml/min.
Detection : spectrophotometer at 276 nm.
Equilibration: before using a new column for
chromatographic analysis, equilibrate by repeated injections
of an insulin solution containing high molecular mass
proteins. This can be done by at least 3 injections of the
resolution solution. The column is equilibrated when
repeatable results are obtained from 2 subsequent injections.
Injection : 100 l.
Run time : about 35 min.
Retention time : polymeric insulin complexes = 13-17 min ;
covalent insulin dimer = about 17.5 min ; insulin
monomer = about 20 min ; salts = about 22 min.
System suitability : resolution solution :
peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to the dimer and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to the
monomer.
Limits : the sum of the areas of any peaks with a retention
time less than that of the principal peak is not greater than
1.0 per cent of the total area of the peaks. Disregard any
peak with a retention time greater than that of the peak due
to insulin.
Related proteins. Liquid chromatography (2.2.29) as
described under Assay, following the elution conditions as
described below :
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 - 30
42
58
30 - 44
42 11
58 89
44 - 50
11
89
01/2005:0831
TESTS
pH (2.2.3). The pH of the suspension to be examined is
6.6 to 7.2.
linearity : inject 20 l each of reference solutions (a)
Insulin in the supernatant : 22.0 per cent to 28.0 per cent of
and (d). The test is not valid unless the area of the
insulin in solution. Determine by the method described in
principal peak in the chromatogram obtained with
the test for insulin in the supernatant in the monograph on
reference solution (a) is 10 0.5 times the area of the
Insulin preparations, injectable (0854).
principal peak in the chromatogram obtained with
reference solution (d). If this test fails, adjust the injection Total zinc : 26.0 g to 37.5 g per 100 IU of insulin.
volume to between 10 l and 20 l, in order that the
Determine by the method described in the monograph on
responses are within the linearity range of the detector.
Insulin preparations, injectable (0854).
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